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• W2907366162 abstract "•An epigenomic roadmap for initiation of X chromosome inactivation (XCI)•Histone deacetylation and H2A ubiquitination are among the earliest XCI events•HDAC3-mediated histone deacetylation is required for efficient XCI•PcG marks are first deposited intergenically and spread when gene silencing occurs During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing. During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing. Successful development requires the establishment and maintenance of euchromatin and heterochromatin in different parts of the genome. Progressively stable silencing of some genic regions occurs as epigenetic memory continues to accrue, finally leading to facultative heterochromatin formation (Trojer and Reinberg, 2007Trojer P. Reinberg D. Facultative heterochromatin: is there a distinctive molecular signature?.Mol. Cell. 2007; 28: 1-13Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Multiple layers of chromatin modifications are believed to enable stable transcriptional silencing. However, little is known about how facultative heterochromatin is dynamically formed and to what extent chromatin changes are involved in the establishment of gene silencing. A powerful model for developmentally induced gene silencing and formation of facultative heterochromatin is X chromosome inactivation (XCI) in female mammals. Although the role for chromatin changes in maintenance of the inactive state has been extensively studied (Disteche and Berletch, 2015Disteche C.M. Berletch J.B. X-chromosome inactivation and escape.J. Genet. 2015; 94: 591-599Crossref PubMed Scopus (72) Google Scholar), almost nothing is known about their role in the initiation of gene silencing. In female mouse embryos, one of the two X chromosomes is randomly chosen for inactivation around the time of implantation (Lyon, 1962Lyon M.F. Sex chromatin and gene action in the mammalian X-chromosome.Am. J. Hum. Genet. 1962; 14: 135-148PubMed Google Scholar). This phenomenon is dependent on the coating of the future inactive X chromosome (Xi) by the long non-coding RNA Xist (Penny et al., 1996Penny G.D. Kay G.F. Sheardown S.A. Rastan S. Brockdorff N. Requirement for Xist in X chromosome inactivation.Nature. 1996; 379: 131-137Crossref PubMed Scopus (1012) Google Scholar). The conserved A-repeat region of Xist mediates transcriptional silencing (Wutz et al., 2002Wutz A. Rasmussen T.P. Jaenisch R. Chromosomal silencing and localization are mediated by different domains of Xist RNA.Nat. Genet. 2002; 30: 167-174Crossref PubMed Scopus (575) Google Scholar), whereas other parts of Xist ensure chromosome coating (Almeida et al., 2017Almeida M. Pintacuda G. Masui O. Koseki Y. Gdula M. Cerase A. Brown D. Mould A. Innocent C. Nakayama M. et al.PCGF3/5-PRC1 initiates Polycomb recruitment in X chromosome inactivation.Science. 2017; 356: 1081-1084Crossref PubMed Scopus (153) Google Scholar, Wutz et al., 2002Wutz A. Rasmussen T.P. Jaenisch R. Chromosomal silencing and localization are mediated by different domains of Xist RNA.Nat. Genet. 2002; 30: 167-174Crossref PubMed Scopus (575) Google Scholar). Assays coupling immunofluorescence with RNA fluorescence in situ hybridization (IF/RNA FISH) have revealed that, upon Xist RNA coating, a program of striking chromatin rearrangements ensues. These include rapid loss of histone modifications associated with active promoters (H3K4me3, H4ac, H3K9ac, and H3K27ac) and enhancers (H3K27ac and H3K4me1) (Chaumeil et al., 2002Chaumeil J. Okamoto I. Guggiari M. Heard E. Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells.Cytogenet. Genome Res. 2002; 99: 75-84Crossref PubMed Scopus (85) Google Scholar, Jeppesen and Turner, 1993Jeppesen P. Turner B.M. The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression.Cell. 1993; 74: 281-289Abstract Full Text PDF PubMed Scopus (598) Google Scholar). Furthermore, there is accumulation of H2AK119Ub and H3K27me3, two repressive histone marks dependent on the activity of Polycomb repressive complex (PRC) 1 and 2, respectively (for a review, see Brockdorff, 2017Brockdorff N. Polycomb complexes in X chromosome inactivation.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 372: 20170021Crossref PubMed Scopus (42) Google Scholar). These progressive alterations of chromatin states are associated with stable repression of the majority of genes on the Xi. Not all X-linked loci are affected in the same way, however, with some genes being silenced much faster than others or even entirely resisting repression (“escapees”) (Disteche and Berletch, 2015Disteche C.M. Berletch J.B. X-chromosome inactivation and escape.J. Genet. 2015; 94: 591-599Crossref PubMed Scopus (72) Google Scholar). The reason for this striking diversity in gene inactivation dynamics remains unclear. It is likely that the susceptibility of loci to Xist spreading plays a role in this process (Borensztein et al., 2017Borensztein M. Syx L. Ancelin K. Diabangouaya P. Picard C. Liu T. Liang J.B. Vassilev I. Galupa R. Servant N. et al.Xist-dependent imprinted X inactivation and the early developmental consequences of its failure.Nat. Struct. Mol. Biol. 2017; 24: 226-233Crossref PubMed Scopus (85) Google Scholar, Engreitz et al., 2013Engreitz J.M. Pandya-Jones A. McDonel P. Shishkin A. Sirokman K. Surka C. Kadri S. Xing J. Goren A. Lander E.S. et al.The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome.Science. 2013; 341: 1237973Crossref PubMed Scopus (682) Google Scholar); however, differences in their chromatin status could also underpin transcriptional silencing dynamics. Although some chromatin marks have previously been mapped on the X chromosome in female embryonic stem cells (ESCs) and differentiated cells, nothing is known about the dynamics of the XCI process, especially during early stages when gene silencing actually takes place (Marks et al., 2009Marks H. Chow J.C. Denissov S. Françoijs K.J. Brockdorff N. Heard E. Stunnenberg H.G. High-resolution analysis of epigenetic changes associated with X inactivation.Genome Res. 2009; 19: 1361-1373Crossref PubMed Scopus (110) Google Scholar, Pinter et al., 2012Pinter S.F. Sadreyev R.I. Yildirim E. Jeon Y. Ohsumi T.K. Borowsky M. Lee J.T. Spreading of X chromosome inactivation via a hierarchy of defined Polycomb stations.Genome Res. 2012; 22: 1864-1876Crossref PubMed Scopus (124) Google Scholar). Specifically, the order of chromatin changes and their possible role(s) in mediating transcriptional silencing remain largely unknown. Previous studies have relied on ESC differentiation for XCI initiation, which results in highly asynchronous Xist induction and elevated levels of heterogeneity in the population. These complications entirely mask primary chromatin events occurring in a small subset of cells embarking on the process of XCI. Recent advances in the identification of Xist binding proteins revealed that most histone-modifying activities are not directly recruited by Xist RNA but, rather, by its binding partners, like SPEN and HNRNPK (Chu et al., 2015Chu C. Zhang Q.C. da Rocha S.T. Flynn R.A. Bharadwaj M. Calabrese J.M. Magnuson T. Heard E. Chang H.Y. Systematic discovery of Xist RNA binding proteins.Cell. 2015; 161: 404-416Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar, McHugh et al., 2015McHugh C.A. Chen C.K. Chow A. Surka C.F. Tran C. McDonel P. Pandya-Jones A. Blanco M. Burghard C. Moradian A. et al.The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.Nature. 2015; 521: 232-236Crossref PubMed Scopus (729) Google Scholar, Monfort et al., 2015Monfort A. Di Minin G. Postlmayr A. Freimann R. Arieti F. Thore S. Wutz A. Identification of Spen as a Crucial Factor for Xist Function through Forward Genetic Screening in Haploid Embryonic Stem Cells.Cell Rep. 2015; 12: 554-561Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, Pintacuda et al., 2017Pintacuda G. Wei G. Roustan C. Kirmizitas B.A. Solcan N. Cerase A. Castello A. Mohammed S. Moindrot B. Nesterova T.B. et al.hnRNPK Recruits PCGF3/5-PRC1 to the Xist RNA B-Repeat to Establish Polycomb-Mediated Chromosomal Silencing.Mol. Cell. 2017; 68: 955-969.e10Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). A major question is whether the chromatin changes that occur early on in XCI are actually involved in gene silencing during XCI and, if so, how. Here we investigate the earliest events accompanying transcriptional silencing of the X chromosome. Using a female ESC line in which one X chromosome can be specifically inactivated via an inducible Xist gene, we performed allele-specific native chromatin immunoprecipitation sequencing (ChIP-seq) as well as nascent transcript profiling at the initiation stages of XCI. Using 4-hr time resolution, we were able to reveal the precise order of chromosome-wide epigenetic events that are intricately coupled to gene repression during XCI. We find that loss of histone acetylation and, in particular H3K27ac, is one of the first events following Xist RNA accumulation during initiation of XCI. We also show that histone deacetylation, via HDAC3, is vital for efficient silencing of most genes on the Xist-coated X chromosome. Contrary to the prevailing hypothesis, HDAC3 is not acutely recruited to the X chromosome by Xist; rather, it is pre-loaded, mainly at putative enhancers. Upon Xist coating, pre-loaded HDAC3 likely mediates histone deacetylation and facilitates transcriptional silencing. We also uncover a surprisingly rapid accumulation of the PRC1-dependent H2AK119Ub mark on the X chromosome, particularly at intergenic regions lying in proximity to Xist RNA entry sites, which are pre-marked by Polycomb (PcG) marks. Accumulation of H3K27me3 appears slightly later and is delayed compared with gene silencing. Using Hdac3 and Xist mutant cells, we also show that spread of Polycomb marks into gene bodies can only occur when transcriptional repression begins. Our study reveals the chromatin choreography across the X chromosome during the early steps of XCI initiation and uncovers a role for histone deacetylation in the establishment of gene silencing. A molecular roadmap of the earliest chromatin changes during the initiation of XCI has not yet been established. To reduce heterogeneity and obtain a highly resolved time series of chromatin events upon Xist RNA coating, we used the TX1072 ESC line (Figure 1A; Schulz et al., 2014Schulz E.G. Meisig J. Nakamura T. Okamoto I. Sieber A. Picard C. Borensztein M. Saitou M. Blüthgen N. Heard E. The two active X chromosomes in female ESCs block exit from the pluripotent state by modulating the ESC signaling network.Cell Stem Cell. 2014; 14: 203-216Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). This hybrid (Mus musculus castaneus x C57BL/6) line harbors a doxycycline (DOX) inducible promoter upstream of the endogenous Xist at the C57BL/6 (B6) allele (Schulz et al., 2014Schulz E.G. Meisig J. Nakamura T. Okamoto I. Sieber A. Picard C. Borensztein M. Saitou M. Blüthgen N. Heard E. The two active X chromosomes in female ESCs block exit from the pluripotent state by modulating the ESC signaling network.Cell Stem Cell. 2014; 14: 203-216Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, Wutz et al., 2002Wutz A. Rasmussen T.P. Jaenisch R. Chromosomal silencing and localization are mediated by different domains of Xist RNA.Nat. Genet. 2002; 30: 167-174Crossref PubMed Scopus (575) Google Scholar). By adding DOX, we synchronously induce Xist expression and can thus decouple the onset of XCI from differentiation-dependent chromatin changes (Figure S1C). We performed allele-specific native ChIP-seq (nChIP-seq) for seven histone modifications across five time points at up to 4-hr resolution on biological duplicates (Figure 1A; Figures S1A and S1B). We focused on histone modifications associated with active promoters (H3K4me3, H3K9ac, H4ac, and H3K27ac) and active enhancers (H3K4me1 and H3K27ac) as well as repressive Polycomb-dependent marks (H3K27me3 and H2AK119Ub). All nChIP-seq datasets were sequenced to a depth of at least 40 million reads and showed a high signal-to-noise ratio (Figures S1B and S1E). The reads were split according to content of allele-specific SNPs (B6, mapping to Xi; Cast, mapping to active X); this allelic information was then analyzed in detail to define XCI-specific changes.Figure S1Allele-Specific Native ChIP-Seq Monitors Changes in Active Histone Modifications during XCI, Related to Figure 1Show full caption(A) nChIP enrichment validation using qPCR. Shown is the enrichment of nChIP signal at positive (color) and negative (black) control regions for each mark individually. Shown is a representative result for one biological replicate. Values were normalized to the input sample. (B) Average enrichment plots for active histone modifications across all transcriptional start sites. (C) RNA FISH quantification of Xist induction (p510 probe) during both replicates of nChIP-seq time course. At least 70 nuclei were quantified for each sample. (D) Bar plots showing the percentage of B6(Xi) reads mapping to the X chromosome in the nChIP-seq time course. (E) Genome browser plots of active histone modification at the Gda3/Dppa3/Nanog cluster. Shown are all mapped reads at all time points in biological duplicates. Note highly reproducible and stable pattern of enrichment. (F) Example showcasing the differences between IC35 and ED50 parameters. The IC35 measures when the loss of a histone mark reaches an efficiency threshold and not the timing when this process occurs most rapidly. For example, if two marks show different efficiency of loss from the Xi (i.e., different IC35) the process might still be occurring at the same time. Shown is H3K27 deacetylation dynamics for two promoters (Fmr1:red, Pgk1:blue). Both promoters have the same ED50 (i.e., time when sigmoid reaches its maximum slope), however show very different efficiency of deacetylation. The latter difference is captured by the IC35 parameter (i.e., time when sigmoid reaches the 0.35 threshold). (G) Heatmaps showing the d-scores of all peaks on the X chromosome. Windows were sorted from centromere (top) to telomere (bottom). (H-I) Plots showing the distribution of differential IC35 (H): H3K4me3-H3K27ac; (I): H3K4me1-H3K27ac) for all promoters (H) and putative enhancers (I). Each bar represents a single window, these were ordered accordingly to their differential IC35. (J) Violin plots comparing the dynamics of H3K27ac and H3K4 methylation loss at active promoters (left) and putative active enhancers (right). Plots compare the ED50 parameter of H3K27ac and H3K4me peaks within the quantitative range of the experiment (IC35 < 24hrs). p value was calculated using paired Wilcoxon rank sum test.(K-L) Violin plots comparing the distribution of IC35 for H3K27ac (K) or H3K4me1/3 (L) at enhancers and promoters. p values calculated using Wilcoxon rank sum test. (M) Plots showing the distribution of differential IC35 for H3K27ac at promoter-putative enhancer pairs. Windows within one TAD were paired based on proximity. Individual pairs were ordered accordingly to their differential IC35. (N) Average H3K4me1 distribution plots around the transcriptional start site of active genes (TSS ± 1kb). Plots were done using all mapped reads on TSS of active genes on X chromosome (top) and on autosomes (bottom). Average profiles show duplicates at 0hr (gray) and 12hrs (orange) of DOX treatment. (O) Boxplots showing the d-score distribution for H3K4me1 around TSS of active genes (±200bp) after 0 (gray) and 12hrs (orange) of DOX treatment, on X chromosome (top) and on autosomes (bottom). p values calculated using Wilcoxon rank sum test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) nChIP enrichment validation using qPCR. Shown is the enrichment of nChIP signal at positive (color) and negative (black) control regions for each mark individually. Shown is a representative result for one biological replicate. Values were normalized to the input sample. (B) Average enrichment plots for active histone modifications across all transcriptional start sites. (C) RNA FISH quantification of Xist induction (p510 probe) during both replicates of nChIP-seq time course. At least 70 nuclei were quantified for each sample. (D) Bar plots showing the percentage of B6(Xi) reads mapping to the X chromosome in the nChIP-seq time course. (E) Genome browser plots of active histone modification at the Gda3/Dppa3/Nanog cluster. Shown are all mapped reads at all time points in biological duplicates. Note highly reproducible and stable pattern of enrichment. (F) Example showcasing the differences between IC35 and ED50 parameters. The IC35 measures when the loss of a histone mark reaches an efficiency threshold and not the timing when this process occurs most rapidly. For example, if two marks show different efficiency of loss from the Xi (i.e., different IC35) the process might still be occurring at the same time. Shown is H3K27 deacetylation dynamics for two promoters (Fmr1:red, Pgk1:blue). Both promoters have the same ED50 (i.e., time when sigmoid reaches its maximum slope), however show very different efficiency of deacetylation. The latter difference is captured by the IC35 parameter (i.e., time when sigmoid reaches the 0.35 threshold). (G) Heatmaps showing the d-scores of all peaks on the X chromosome. Windows were sorted from centromere (top) to telomere (bottom). (H-I) Plots showing the distribution of differential IC35 (H): H3K4me3-H3K27ac; (I): H3K4me1-H3K27ac) for all promoters (H) and putative enhancers (I). Each bar represents a single window, these were ordered accordingly to their differential IC35. (J) Violin plots comparing the dynamics of H3K27ac and H3K4 methylation loss at active promoters (left) and putative active enhancers (right). Plots compare the ED50 parameter of H3K27ac and H3K4me peaks within the quantitative range of the experiment (IC35 < 24hrs). p value was calculated using paired Wilcoxon rank sum test.(K-L) Violin plots comparing the distribution of IC35 for H3K27ac (K) or H3K4me1/3 (L) at enhancers and promoters. p values calculated using Wilcoxon rank sum test. (M) Plots showing the distribution of differential IC35 for H3K27ac at promoter-putative enhancer pairs. Windows within one TAD were paired based on proximity. Individual pairs were ordered accordingly to their differential IC35. (N) Average H3K4me1 distribution plots around the transcriptional start site of active genes (TSS ± 1kb). Plots were done using all mapped reads on TSS of active genes on X chromosome (top) and on autosomes (bottom). Average profiles show duplicates at 0hr (gray) and 12hrs (orange) of DOX treatment. (O) Boxplots showing the d-score distribution for H3K4me1 around TSS of active genes (±200bp) after 0 (gray) and 12hrs (orange) of DOX treatment, on X chromosome (top) and on autosomes (bottom). p values calculated using Wilcoxon rank sum test. Upon Xist coating, the Xi becomes rapidly depleted of active histone modifications. Although this global loss of euchromatin occurs in a similar time window as the changes in X-linked transcriptional activity (Chaumeil et al., 2002Chaumeil J. Okamoto I. Guggiari M. Heard E. Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells.Cytogenet. Genome Res. 2002; 99: 75-84Crossref PubMed Scopus (85) Google Scholar), the precise chronology and possible mechanistic relationship of these two events have remained unclear. To address this, we first analyzed nChIP-seq datasets for a panel of active histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K9ac, and H4ac; Figure S1E). This revealed progressive depletion of B6-specific reads (originating from Xi) upon Xist induction, with different marks showing different kinetics of loss (Figure S1D). To analyze this in detail, we performed peak calling on all the reads (STAR Methods). For each peak with at least 50 allele-specific reads, we calculated its d-score (readsB6/[readsCast + readsB6]). This parameter of allelic skewing uses the active X chromosome as a powerful internal control, rendering the analysis less sensitive to variability in nChIP-seq quality and, thus, more stable between replicates. We first set out to track the relative dynamics of active chromatin marks by comparing d-score dynamics of all peaks. We found that loss of H3K27ac and H4ac is among the earliest and most robust chromatin events on the X chromosome following Xist induction (Figure 1B). Furthermore, we found that not only did different histone modifications show distinct behaviors but, also, that specific regions of the X chromosome followed rather different dynamics of inactivation (Figure S1G). Thus, there is a temporal hierarchy of chromatin events both at the level of different histone marks and genomic loci. To better understand in which context chromatin changes might affect gene silencing, we separately focused on promoters and putative enhancers. For each window, we plotted a d-score (normalized to t = 0 hr) as a function of time and fitted a sigmoidal curve (STAR Methods). We found that the maximal dynamic range of inactivation was from 0.5 to ∼0.2 and, thus, set a threshold for when the curve reached 50% of this dynamic range; i.e., d = 0.35 (STAR Methods). This IC35 (inactivation criterium 0.35) parameter indicates how quickly a given peak becomes significantly decreased or lost (Figure S1F). Pairwise comparisons of the IC35 at X-linked gene promoters revealed that H3K27ac becomes efficiently deacetylated prior to demethylation of H3K4me3 (Figure 1C). This trend holds true for ∼88% of measured promoters (Figure S1H), including a representative example of the Pdk3 promoter (Figure 1D). Similar analysis of the enhancer inactivation dynamics revealed that H3K27ac is efficiently depleted prior to loss of H3K4me1 (Figure 1C). This robust difference is observed in ∼95% of the cases; e.g., at a putative Pdk3 enhancer element (Figure 1E; Figure S1I). Thus, histone deacetylation is an early and efficient event during XCI. Next, to extract information about relative timing rather than the efficiency threshold, we obtained the time when the curve reaches its maximum slope (effective dose 50%, ED50; Figure S1F). ED50 analysis revealed that both H3K27ac and H3K4me3 become depleted concomitantly (Figure S1J). Together with the IC35 data, this indicates that, even though demethylation of promoters and their deacetylation are concurrent, the latter process is significantly more efficient. Furthermore, unlike promoters, enhancers showed significant differences in the ED50 parameter between H3K27ac and H3K4me1, indicating that enhancer demethylation is not only less efficient but also significantly delayed (Figure S1J). To assess the relative deacetylation dynamics of enhancers and promoters, we compared the IC35 parameter for H3K27ac at both types of genomic regulatory elements. Intriguingly, enhancer deacetylation was slightly more efficient than that of promoters (Figure S1K). After pairing active promoters to their putative enhancers (STAR Methods), ∼76% (37 of 49) of them showed more efficient deacetylation at enhancers than at promoters (Figure S1M). On the other hand, loss of H3K4me1 on the Xi, which is indicative of enhancer decommissioning, was found to be significantly less efficient compared with the loss of H3K4me3 at promoters (Figure S1L). Thus, inactivation (loss of H3K27ac) of enhancers proceeds at a slightly higher efficiency than of promoters, although stable decommissioning (loss of H3K4me) of promoters precedes that of enhancers. Intriguingly, upon H3K4me3 loss following Xist induction, we observe a slight increase in H3K4me1 levels around transcriptional start sites (TSSs), which probably reflects an intermediate of stepwise enzymatic H3K4me3 demethylation (Figures S1N and S1O). In summary, our data show that efficient histone deacetylation at promoters and enhancers is one of the first events during XCI, indicating that it might contribute to initiation of transcriptional silencing. In comparison, demethylation of H3K4me3 and H3K4me1 is less efficient and delayed, respectively. Finally, deacetylation of promoters and enhancers seems to follow broadly comparable dynamics, whereas their decommissioning differs significantly. To gain insight into the relationship between loss of active chromatin marks and transcriptional silencing of X-linked genes, we compared our nChIP-seq dataset with transient transcriptome sequencing (TT-seq; Schwalb et al., 2016Schwalb B. Michel M. Zacher B. Frühauf K. Demel C. Tresch A. Gagneur J. Cramer P. TT-seq maps the human transient transcriptome.Science. 2016; 352: 1225-1228Crossref PubMed Scopus (232) Google Scholar). This allows a direct measure of newly synthesized RNA and, thus, enabled us to measure transcriptional activity as the X chromosome becomes inactivated. Comparison of the IC35 for TT-seq and individual histone modifications at associated promoters revealed that transcriptional silencing dynamics are most tightly linked to promoter deacetylation at H3K27 and, to a lesser extent, to loss of H4ac, H3K9ac, and H3K4me3 (Figures 2A and 2B ; Figure S2A). A similar pattern of gene silencing and promoter deacetylation dynamics further exemplifies this correlation (Figure 2C).Figure S2Histone Deacetylation Correlates with Transcriptional Silencing and Some Genomic Features, Related to Figure 2Show full caption(A) Violin plots comparing the dynamics of transcriptional silencing and the loss of active histone modifications from associated promoters. Plots compare the IC35 parameter calculated from TT-seq nascent transcription profiling to the IC35 of different active histone marks (H3K27ac, H4ac, H3K9ac, H3K4me3). p values were calculated using paired Wilcoxon rank sum test. (B) Heatmap of H3K27ac allelic dynamics at all promoters clustered using k-means. (C) An array showing features enriched (gray font) or depleted (black font) at promoters efficiently inactivated (low IC35) compared to late inactivated (higher IC35) using a panel of histone modifications. Shown are p values calculated using Wilcoxon rank sum test with Benjamini Hochberg correction. Red cells are reaching statistical significance of p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Violin plots comparing the dynamics of transcriptional silencing and the loss of active histone modifications from associated promoters. Plots compare the IC35 parameter calculated from TT-seq nascent transcription profiling to the IC35 of different active histone marks (H3K27ac, H4ac, H3K9ac, H3K4me3). p values were calculated using paired Wilcoxon rank sum test. (B) Heatmap of H3K27ac allelic dynamics at all promoters clustered using k-means. (C) An array showing features enriched (gray font) or depleted (black font) at promoters efficiently inactivated (low IC35) compared to late inactivated (higher IC35) using a panel of histone modifications. Shown are p values calculated using Wilcoxon rank sum test with Benjamini Hochberg correction. Red cells are reaching statistical significance of p < 0.05. The striking variability in dynamics of promoter chromatin changes begged the question of whether these differences could be due to transcriptional status or genomic context; for example, proximity to Xist (the locus and/or Xist RNAs entry" @default.
• W2907366162 created "2019-01-11" @default.
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• W2907366162 date "2019-01-01" @default.
• W2907366162 modified "2023-10-16" @default.
• W2907366162 title "The Implication of Early Chromatin Changes in X Chromosome Inactivation" @default.
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